Refolding Record:
| Protein | |
|---|---|
| Protein Name | Enteropeptidase |
| Abbreviated Name | Enteropeptidase |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P98073 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Heterodimer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | catalytic subunit |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 235 |
| Molecular Weight | 26254.8 |
| Pi | 5.73 |
| Molecular Weight | 26254.8 |
| Disulphides | 5 |
| Full Sequence |
IVGGSNAKEGAWPWVVGLYYGGRLLCGASLVSSDWLVSAAHCVYGRNLEPSKWTAILGLHMKSNLTSPQTVPRLIDEIVINPHYNRRRKDNDIAMMHLEFKVNYTDYIQPICLPEENQVFPPGRNCSIAGWGTVVYQGTTANILQEADVPLLSNERCQQQMPEYNITENMICAGYEEGGIDSCQGDSGGPLMCQENNRWFLAGVTSFGYKCALPNRPGVYARVSRFTEWIQSFLH
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Gasparian ME, Ostapchenko VG, Dolgikh DA, Kirpichnikov MP. (2006) Biochemistry (Moscow), 71, 113-119 |
| Project Aim | Identification and Characterization |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 23.0 |
| Expression Time | 28 h |
| Expression Vector | pET32a |
| Expression Protocol | Escherichia coli BL21(DE3) cells were transformed by plasmid pET-32a/L-HEP. Expression, renaturation, and purification of L-HEP were done as described in our previous paper [16][Escherichia coli BL21(DE3) was transformed by plasmid pET-32a/L-HEP. One colony (2 mm in diameter) was inoculated into 10 mL LB supplemented with 100 μg mL−1 ampicillin and grown overnight at 37 °C. The culture was diluted with 100 mL TB (with 100 μg mL−1 ampicillin) to reach a cell density of 0.1 (absorbance at 550 nm) and grown for 2–2.5 h at 37 °C up to a cell density of 0.7. IPTG was added to a final concentration of 0.07 mM and cultivation was continued for 23 h at 28 °C. ] |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | French Press |
| Lytic Agent | Detergents |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 0.1 M Tris–HCl (pH 8.6), 1 mM EDTA-Na, 20 mM dithiothreitol, and 6 M guanidine–HCl |
| Refolding Buffer | 600 mL of 0.7 M arginine–HCl (pH 8.6), 2 mM reduced glutathione, and 1 mM EDTA |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no |
| Refolding pH | 8.6 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 75 h |
| Redox Agent | GSH |
| Redox Agent Concentration | 2 mM |
| Refolding Protocol | The cells were collected by centrifugation at 5000g for 10 min, 4 °C, resuspended in 37 mL of buffer containing 20 mM Tris–HCl (pH 7.5), 10 mM EDTA, 1% Triton X-100, and lysed in French press. Inclusion bodies were sedimented at 50 000g for 20 min, 4 °C. The protein (Trx/L-HEP) was solubilized at room temperature in 10 mL of 0.1 M Tris–HCl (pH 8.6), 1 mM EDTA-Na, 20 mM dithiothreitol, and 6 M guanidine–HCl. L-HEP was refolded by a modification of a protocol for bovine enteropeptidase light chain [24]. Insoluble material was removed by centrifugation at 80 000g for 20 min, 4 °C. The solubilized protein was dialyzed at room temperature against 3 M guanidine–HCl (pH 2.5), mixed with 10 mL of oxidation buffer (50 mM Tris–HCl, pH 8.3, 6 M guanidine–HCl, and 0.1 M oxidized glutathione), and dialyzed against 3 M guanidine–HCl (pH 8.0). To initiate disulfide exchange and refolding, the protein solution was dropwise diluted in 600 mL of 0.7 M arginine–HCl (pH 8.6), 2 mM reduced glutathione, and 1 mM EDTA, and incubated for 75 h at 4 °C. After dialyzing the reaction mixture for 2 h against 0.1 M Tris–HCl (pH 8.0) containing 1 M CaCl2 at room temperature, complete autocatalytic cleavage of fusion protein took place. NaCl to final concentration of 500 mM was added to the dialyzed protein and active L-HEP was purified by affinity chromatography on STI-agarose. Two milliliters of STI agarose was equilibrated with buffer containing 10 mM Tris–HCl (pH 8.0), 500 M NaCl, and the protein solution was applied at 1 mL min−1 at room temperature. The column was washed with 10 mL of 10 mM Tris–HCl, 500 mM NaCl (pH 8.0). The active L-HEP was eluted with 75 mM glycine–HCl (pH 3.0), 500 mM NaCl; 1 mL fractions were collected and neutralized immediately with 50 μL of 2 M Tris–HCl (pH 8.0). The purified protein was stored in buffer containing 0.1 M Tris–HCl (pH 8.0), 50% glycerol, and 500 mM NaCl at −20 °C. The N-terminal amino acid sequence of L-HEP was determined after SDS–PAGE and electroblotting into a polyvinylidene difluoride (PVDF) membrane [25]. The concentration of L-HEP was determined spectrophotometrically by absorbance at 280 nm using a molar extinction coefficient of 54,450 M−1 cm−1 calculated from the known amino acid sequence [26]. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 2,0% |
| Purity | n/a |
| Notes | The information for this page brought up from Gasparian, M. E., Ostapchenko, V. G., Schulga, A. A., Dolgikh, D. A., and Kirpichnikov, M. P. (2003) Protein. Exp. Purif., 31, 133-139. |